Method of monitoring thrombus formation

ABSTRACT

A method of monitoring thrombus formation is described, which comprises flowing anticoagulated blood into a thrombus formation chamber, in at least a part of which a thrombus formation inducer inducing thrombus formation is provided, while releasing the anticoagulant treatment or promoting blood coagulation to thereby monitor thrombus formation in the thrombus formation chamber.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of and claims the priority benefit ofU.S. application Ser. No. 12/083,787, filed on Aug. 28, 2009, nowallowed, which is a 371 of international application of PCT applicationserial no. PCT/JP2006/320789, filed on Oct. 18, 2006, which claims thepriority benefit of Japan application no. 2005-302557, filed on Oct. 18,2005, Japan application no. 2005-308065, filed on Oct. 24, 2005, Japanapplication no. 2005-334594, filed on Nov. 18, 2005, Japan applicationno. 2005-358448, filed on Dec. 13, 2005, Japan application no.2006-036148, filed on Feb. 14, 2006 and Japan application no.2006-234270, filed on Aug. 30, 2006. The entirety of each of theabove-mentioned patent applications is hereby incorporated by referenceherein and made a part of this specification.

TECHNICAL FIELD

The present invention relates to a method of monitoring efficacy of anantithrombotic drug administered to a patient or the like, andspecifically, to an apparatus for and a method of comprehensivelyevaluating blood coagulation and platelet thrombus formation under abloodstream-equivalent environment with a whole blood or plasmacontaining platelets.

BACKGROUND ART

For example, the atherothrombosis such as the myocardial infarctioncauses serious thrombus formation such that an atheromatous plaque isbroken at an arteriosclerosis site, platelets are adhered on collagenincluding tissue factor exposed to the bloodstream. Further, theplatelet aggregation, the activation of a blood coagulation system, andthe like complexly occur resulting in serious obstructive thrombus.Heart disease such as myocardial infarction, is a serious disease and isthe second leading cause of overall deaths in Japan.

However, thrombus formation proceeds only in an atherosclerotic regionin the myocardial infarction, and a thrombotic tendency in the wholebody is not extremely proceeded. In-vitro examinations are unsuitablefor evaluating the thrombotic tendency in such a thrombosis and themonitoring of the antithrombotic effect in the antithrombotic therapy.Thus, it is important to make comprehensive evaluations on coagulationand platelets (adhesion and agglutination) in the presence of thebloodstream.

Heretofore, the blood coagulability has been evaluated by determiningactivated partial thromboplastin time (APTT), thromboplastin time (PT)using the plasma. The APTT mainly reflects intrinsic coagulation and thePT mainly reflects extrinsic coagulation. The examination of bloodplatelets is carried out by using platelet-rich plasma and adding aplatelet-activating substance such as ADP or collagen to therebyevaluate the aggregating property of platelets from a change intransmittance thereof or the like. In addition, the coagulation time ofthe whole blood can be determined with the whole blood clotting time,the whole blood clotting time after calcium re-addition, and the like.

Further, an examination system using the whole blood employsthromboelastogrm, which monitors the activations of clotting factors,the platelet agglutination, and the like.

However, thrombus grows under a blood flow in vivo. In contrast, theabove examination method or the like is determined in-vitro that is inthe closed state. Thus, the status of in-vivo thrombus growth cannot beobserved.

As proposals for solving the above problems, Patent Document 1 andNon-patent Documents 2 and 3 disclose the method including bringing theblood provided with an antithrombotic drug to be evaluated to pass on acollagen cell and monitoring the adhesion or agglutination of theplatelets by fluorescently-labeling the platelets with a confocalmicroscope.

However, in the invention described in the document the observation iscarried out under the presence of an anticoagulation drug. Thus, thefact that a thrombus which is caused by the adhesion or agglutination ofplatelets induced by the blood coagulation system is not formed ordecreased property to form thrombus is evaluated by monitoring amorphological change in platelet. Thus, the evaluation does not reflectthe platelet activation interlocking with coagulation system. Therefore,such an invention is favorable for the evaluation of the efficacy of anantiplatelet drug but is unable to monitor a thrombus itself and thewhole process of thrombus formation. In addition, a fluorescencemicroscope is expensive, so it can be hardly used for generalexamination.

Further, in Patent Document 2, the fluidity of the anticoagulated bloodis determined by passing the blood through a fine-comb-like siliconcell. Likewise, the process of Patent Document 2 also uses theanticoagulated blood, so the influence of a coagulation system cannot bedetermined. In addition, the viscosity of blood in the process has largeindividual variations and in diurnal variations, so it is difficult toreflect drug therapy using the system.

The platelet is activated by the coagulation system, and the coagulationsystem is promoted by activated platelets. Therefore, the efficacy of anantithrombotic drug cannot be observed in the anticoagulated blood,because activation of platelet is also suppressed by the anticoagulationtreatment. In addition, non-anticoagulated blood can not used in anexamination, because it promptly forms clot.

-   Patent Document 1: JP 2004-251630 A-   Patent Document 2: JP 2006-145345 A-   Non-patent Document 1: Blood. 1990; 75:390-398-   Non-patent Document 2: Blood. 1999; Aug. 1:94(3):968-75

DISCLOSURE OF THE INVENTION

The present invention has been made in consideration of the abovecircumstances and intends to provide an apparatus and method ofcomprehensively evaluating the thrombus formation due to the bloodcoagulation and platelet under a bloodstream-equivalent environment witha whole blood or plasma containing platelets (in the specification ofthe present invention, they may be inclusively referred to as “blood”),when monitoring the efficacy of an antithrombotic drug administered to apatient or the like.

Means for Solving the Problems

To solve the above-mentioned problems, the present invention provides anapparatus for monitoring thrombus formation, which monitors thrombusformation by flowing anticoagulated blood through a channel thatsimulates a blood vessel while releasing an anticoagulation treatment orpromoting a blood coagulation, comprising: a thrombus formation chamberin at least a part of which a thrombus inducing material that inducesthrombus formation is provided; an inlet tube which is connected to thethrombus formation chamber and through which blood is flown into thethrombus formation chamber; and a drug tube which is connected to theinlet tube and through which a drug that promotes blood coagulation(hereinafter may be referred to as “coagulation promotion”) or a drugthat releases the anticoagulation treatment is supplied. In the presentinvention, the term “monitoring” means not only visual evaluation ofthrombus formation with eyes, an imaging, but also evaluation of thedegree of thrombus formation in numerical terms by pressuredetermination or the like.

Further, the present invention provides an apparatus for monitoringthrombus formation which comprises a thrombus formation chamber in atleast a part of which a thrombus inducing material that induces thrombusformation is provided; an inlet tube which is connected to the thrombusformation chamber and through which blood is flown into the thrombusformation chamber; and a thrombus formation inhibitor inlet tube whichis connected to the thrombus formation chamber and mixes a thrombusformation inhibitor with the blood after passing through the thrombusformation chamber.

In this case, the apparatus for monitoring thrombus formation ispreferably formed on a substrate.

The apparatus for monitoring thrombus formation of the present inventionpreferably further comprises a pump for pressurizing the inlet tubeand/or the drug tube or a pump for aspirating a discharge tube which isconnected to the thrombus formation chamber and provided for dischargingthe blood from the thrombus formation chamber.

The apparatus for monitoring thrombus formation of the present inventionpreferably further include a pressure-measuring apparatus and a camerafor taking images of a thrombus formation chamber.

Further, the thrombus inducing material preferably comprises collagen.

More preferably, the thrombus inducing material further comprises atissue factor (tissue thromboplastin).

Further, the present invention provides a method of monitoring thrombusformation, comprising: flowing anticoagulated blood into a thrombusformation chamber, in at least a part of which a thrombus inducingmaterial inducing thrombus formation is provided, while releasing ananticoagulation treatment or promoting a blood coagulation to therebymonitor thrombus formation. In the present invention, the term “flowingthe blood while releasing the anticoagulation treatment or promotingblood coagulation” may be a state where an anticoagulation-releasingreaction or a coagulation-promoting reaction in the channel is beingoccurred, and includes a state where an drug that releases ananticoagulation releasing agent, or a coagulation promoting agent isflown while mixing with the blood in the channel or a state where theanticoagulation releasing agent or the coagulation promoting agent ispromptly flown after mixing with the blood.

In the method of monitoring thrombus formation of the present invention,it is preferable that the anticoagulation treatment is a treatment witha calcium chelator such as citric acid and the anticoagulation treatmentis released with a free calcium donor.

In the method of monitoring the thrombus formation of the presentinvention, it is preferable that the anticoagulation treatment is atreatment with a thrombin aptamer and the anticoagulation treatment isreleased with the antisense DNA of the thrombin aptamer.

Here, in the method of monitoring thrombus formation of the presentinvention, it is preferable to monitor the thrombus formation by flowinganticoagulated blood into a thrombus formation chamber, while promotingblood coagulation without releasing the anticoagulation treatment. Inthis case, a means for promoting the blood coagulation is preferably theaddition of tissue thromboplastin.

In addition, in the method of monitoring thrombus formation of thepresent invention, the blood which has been anticoagulated with one kindor more kinds of anticoagulation agents is preferably released form theanticoagulation treatment with at least one kind of anticoagulationtreatment releasing agent that corresponds to the anticoagulationtreatment agent used. Here, it is preferable that the anticoagulationtreatment agents are a contact phase factor inhibitor and a calciumchelator, and the anticoagulation treatment releasing agent is a freecalcium donor. Further, it is also preferable that the anticoagulationtreatment agents are a contact phase factor inhibitor and heparin, andthe anticoagulation treatment releasing agent is heparinase. Further, itis also preferable that the anticoagulation treatment agents are aninhibitor for a contact phase factor such as a blood coagulation XIIfactor, kallikrein, or the like and a thrombin aptamer, and theanticoagulation treatment releasing agent is the antisense DNA of thethrombin aptamer. The inhibitor for the blood coagulation XII factor ispreferably a maize-derived trypsin inhibitor.

In the method of monitoring thrombus formation of the present invention,it is preferable to determine the pressure at the time of inflow and/oroutflow of the blood in the thrombus foil cation chamber.

In the method of monitoring thrombus formation of the present invention,the thrombus inducing material preferably comprises collagen and atissue factor.

Effects of the Invention

According to an apparatus for monitoring thrombus formation of thepresent invention, the apparatus comprises: a thrombus formation chamberin at least a part of which a thrombus inducing material that inducesthrombus formation is provided; an inlet tube which is connected to thethrombus formation chamber and through which blood is flown into thethrombus formation chamber; and a drug tube which is connected to theinlet tube and through which a drug that releases the anticoagulationtreatment or a drug that promotes blood coagulation is supplied.Therefore, the anticoagulated blood, which is treated in order toprevent the blood collected after administering the antithrombotic drugto a patient from coagulating in the channel extending to the thrombusformation chamber, can be monitored by intentionally forming a thrombusin a thrombus formation chamber. Thus, the efficacy of an antithromboticdrug can be specifically monitored in the environment similar to theinside of the human body. In addition, an anticoagulation treatmentagent can be used at the time of blood sampling. Therefore, there is anadvantage in that samples after the blood sampling can be stored for acertain period of time and the examination time can be randomlyselected.

According to another apparatus for monitoring thrombus formation of thepresent invention, the apparatus comprises: a thrombus formation chamberin at least a part of which a thrombus inducing material that inducesthrombus formation is provided; an inlet tube which is connected to thethrombus foil cation chamber and through which blood is flown into thethrombus formation chamber; and an inlet for a thrombus formationinhibitor, which is connected to the thrombus formation chamber andmixes the thrombus formation inhibitor with the blood after passingthrough the thrombus formation chamber. Therefore, the thrombusobservation can be carried out in a manner as described above. Inaddition, the blood coagulation does not proceed downstream in thethrombus formation chamber, therefore, influence on the pressuredetermination can be prevented and more delicate pressure changes can bemonitored.

A small amount of blood can be monitored when the apparatus formonitoring thrombus formation of the present invention is formed on asubstrate. In addition, the apparatus is provided with a pump forpressurizing the inlet tube and/or the drug tube or a pump foraspirating a discharge tube which is connected to the thrombus formationchamber and provided for discharging the blood from the thrombusformation chamber. Therefore, the blood and the drug for promoting bloodcoagulation can be stably flown for a predetermined period of time atpredetermined pressure or predetermined flow rate.

If the apparatus for monitoring thrombus formation of the presentinvention comprises a pressure-measuring apparatus, the degree ofthrombus formation can be converted into numbers, so a quantitativeevaluation can be performed.

The apparatus can be easily set when the thrombus inducing materialcomprises collagen. Thrombus formation can be efficiently induced whenthe thrombus inducing material further comprises a tissue factor such astissue thromboplastin together with collagen.

Further, if the apparatus for monitoring thrombus formation of thepresent invention comprises a camera for taking an image of the thrombusformation chamber, the appearance of the thrombus formation can beobserved as an image and the image can be then stored.

Further, according to a method of monitoring thrombus formation of thepresent invention, the method comprises: monitoring thrombus formationby flowing anticoagulated blood through a thrombus formation chamber inat least a part of which a thrombus inducing material that inducesthrombus formation is provided, while releasing the anticoagulationtreatment or promoting blood coagulation. Thus, the thrombus formationon the thrombus inducing material can be monitored by flowing theanticoagulated blood, which is obtained by anticoagulating the bloodcollected after administering the antithrombotic drug to a patient toprevent coagulation, while promoting blood coagulation. Therefore, theefficacy of an antithrombotic drug can be specifically monitored in theenvironment similar to the inside of the human body. In addition, ananticoagulation agent can be used for blood sampling. Therefore, thereis an advantage in that samples after the blood sampling can be storedfor a certain period of time and the examination time can be randomlyselected. If the anticoagulation treatment is a treatment with a calciumchelator such as citric acid and the anticoagulation treatment isreleased by a free calcium donor, the reagent can be easily obtained andthus it is preferable. If the anticoagulation treatment is a treatmentwith a thrombin aptamer and the anticoagulation treatment is released bythe antisense DNA of the thrombin aptamer, it is possible to carry outthe examination while reflecting the physiological calcium ionconcentration of the blood.

In addition, according to another method of monitoring thrombusformation of the present invention, it is able to monitor thrombusformation by flowing anticoagulated blood into the thrombus formationchamber without performing the operation of releasing theanticoagulation treatment, while prompting blood coagulation. Therefore,the thrombus formation can be observed with a small amount of the bloodand the burden of the subject can be reduced. In this case, further, thedrug tube is not always required, so the apparatus for monitoringthrombus formation can be simplified. Here, if tissue thromboplastin isused as a blood coagulation promoting agent, the blood coagulation canbe promoted by activating the coagulation system in an alternativepathway that avoids the XII-factor activation and the kallikreinactivation to thereby monitor thrombus formation in the thrombusformation chamber.

Further, the thrombus formation can be monitored with a simple operationby releasing the anticoagulated blood obtained using one kind or morekinds of anticoagulation treatment agents with at least one kind ofanticoagulation treatment releasing agent corresponding to theanticoagulation treatment agent used. In this case, when ananticoagulation treatment is carried out with a contact phase factorinhibitor and a calcium chelator, and the anticoagulation treatment isreleased with a free calcium donor, or when an anticoagulation treatmentis carried out with a contact phase factor inhibitor, and heparin andthe anticoagulation treatment is released with heparinase, theanticoagulation treatment exerting an effect at the time of monitoringthrombus formation is an anticoagulation treatment with the contactphase factor inhibitor, therefore monitoring of thrombus formation canbe performed under more physiological conditions, particularly whilereflecting a divalent metal ion associated with thrombosis, such ascalcium or magnesium. In this case, when the anticoagulation treatmentis carried out with an inhibitor of a contact phase such as a bloodcoagulation XII factor or kallikrein and a thrombin aptamer, and thenthe anticoagulation treatment is released by the antisense DNA of thethrombin inhibition aptamer, the blood can be stored over a prolongedperiod. Further, the anticoagulation treatment can be efficientlycarried out when a maize-derived trypsin inhibitor is used as theinhibitor of the blood coagulation XII factor.

If the method of monitoring thrombus formation of the present inventionperforms the measurement of pressure at the time of inflow and/oroutflow of the blood in the thrombus formation chamber, the degree ofthrombus formation can be converted into numbers, so a quantitativeevaluation can be easily performed by an extremely simple apparatus.

Further, if the thrombus inducing material comprises collagen and thetissue factor, the apparatus can be easily set and the thrombusformation can be effectively induced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating an apparatus for monitoringthrombus formation according to a first embodiment of the presentinvention.

FIG. 2 (a) is a schematic diagram illustrating installation conditionsof a thrombus inducing material 15 of Examples 3, 5, and 6 according tothe first embodiment of the present invention, and FIG. 2 (b) is aschematic diagram illustrating results of thrombus formation of Examples3, 5, and 6 according to the first embodiment of the present invention.

FIG. 3 (a) is a schematic diagram illustrating installation conditionsof the thrombus inducing material 15 of Example 4 of the firstembodiment of the present invention, and FIG. 3 (b) is a schematicdiagram illustrating results of thrombus formation of Example 4according to the first embodiment of the present invention.

FIG. 4 is a schematic diagram illustrating a main part of an apparatusfor monitoring thrombus formation (main body of a microchip) accordingto a second embodiment of the present invention.

FIG. 5 is a schematic diagram illustrating a main part of the apparatusfor monitoring thrombus formation (cover of a microchip) according tothe second embodiment of the present invention.

FIG. 6 is a schematic diagram illustrating the position of thrombusformation of Example 7 according to the second embodiment of the presentinvention.

FIG. 7 is a schematic diagram illustrating a main part of an apparatusfor monitoring thrombus formation of another example (main body of amicrochip) according to the second embodiment of the present invention.

FIG. 8 is a schematic diagram illustrating a main part of the apparatusfor monitoring thrombus formation (cover of a microchip) of anotherexample according to the second embodiment of the present invention.

FIG. 9 are schematic diagrams illustrating main parts of an apparatusfor monitoring thrombus formation of another example according to athird embodiment of the present invention, where FIG. 9 (A) illustratesa completion drawing, FIG. 9 (B) illustrates a main body of a microchip,and (C) illustrates a cover of the microchip.

FIG. 10 is a schematic diagram illustrating a thrombus-monitoring systemsystemized using the apparatus for monitoring thrombus formation of thepresent invention.

FIG. 11(A) is a result obtained by analyzing a thromboelastogramwaveform of coagulation by adding 10 μM of each of aptamers for exositeI and exosite II to the blood, and after storing the blood at roomtemperature for 15 minutes, adding 40 μM of each of antisense DNAs ofboth aptamer. FIG. 11(B) illustrates the thromboelastogram waveform ofthe blood just after blood sampling.

FIG. 12 are schematic diagrams illustrating main parts of an apparatusfor monitoring thrombus formation according to a fourth embodiment ofthe present invention, where FIG. 12 (A) illustrates a completiondrawing, FIG. 12 (B) illustrates a cover of a microchip, and FIG. 12 (C)illustrates a substrate of the microchip.

FIG. 13 is a graph showing results of pressure measurements of Example17 (control), Example 18 (heparin), and Example 19 (Reopro).

FIG. 14 is a graph showing results of pressure measurements withaddition of 0 (control), 0.2, 0.5, and 1 unit/ml of heparin.

FIG. 15 is a graph showing results of pressure measurements withaddition of 0 (control), 2, 5, and 10 μg/ml of heparin.

DESCRIPTION OF REFERENCE NUMERALS

-   1 . . . apparatus for monitoring thrombus formation,-   10, 110, 311, 411 . . . thrombus formation chamber,-   11, 111 . . . inlet tube,-   12, 112, 312 . . . drug tube,-   13, 313, 413 . . . discharge tube,-   14, 114 . . . constriction portion,-   15, 115, 315 . . . thrombus inducing material,-   16 . . . generated thrombus,-   20, 21, 320, 420 . . . syringe,-   30, 31, 330, 331, 414, 423 . . . pump,-   40, 41, 340 . . . pressure sensor,-   100 . . . substrate (main body of microchip),-   200 . . . substrate (cover of microchip),-   100A, 100B, 100C . . . connection part,-   100D . . . circuit to be pressure gauge,-   100E . . . regulation valve,-   300, 400 . . . microchip (apparatus for monitoring thrombus    formation),-   306 . . . pump control unit,-   307 . . . computer,-   308 . . . fluorescent stereoscopic microscope with CCD camera,-   317 . . . pressure inlet tube,-   322, 422 . . . blood caoagulation inhibitor inlet,-   412 . . . connection tube,-   430 . . . CCD camera,-   431 . . . lens,-   432 . . . illumination optical source,-   433 . . . rail for moving camera,-   A, B . . . thrombus-monitoring system

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described with reference tothe attached drawings in accordance the best mode.

FIG. 1 is a schematic diagram illustrating a first embodiment of theapparatus for monitoring thrombus formation of the present invention.

As shown in FIG. 1, the apparatus 1 for monitoring thrombus formation ofthis embodiment comprises a thrombus formation chamber 10; an inlet tube11 which is connected to the thrombus formation chamber and throughwhich blood is flown into the thrombus formation chamber; and a drugtube 12 which is connected to the inlet tube and through which a drugthat releases the anticoagulation treatment or a drug that promotesblood coagulation is supplied.

The thrombus formation chamber 10 is in the form of a substantiallycylindrical shape provided with a thrombus-inducing material thatinduces thrombus formation in a part of the inside thereof and can beproduced from a transparent glass, a thermoplastic resin, or the like.Examples of the thrombus inducing material include collagen, vWF (vonWillebrand factor), previously-prepared thrombus, and fibrous substratesof silk, cotton, or the like. These materials may be used solely or incombination of two or more thereof. Among them, collagen is particularlypreferable because it can be easily obtained, easily handled, andprovided as a model similar to the actual blood vessel. The collagen maycomprise a tissue factor. The thrombus inducing material of collagen orvWF is preferably in a state of being coated inside of the thrombusformation chamber 10 to prevent the thrombus inducing material fromoutflowing with the blood flow. Coating can be easily performed, forexample, as described in JP 05-260950 A or Blood. 1995 Apr. 1; 85(7):1826-35, by dissolving collagen in an acidic solution and dippingtherein a substrate having hydrophillicity such as glass or polystyrene,followed by washing and drying to coat the surface of the material.

Further, it is preferable that the thrombus inducing material of afibrous material or a previously-prepared thrombus may be in a state ofbeing fixed in the inside of the thrombus formation chamber 10. Further,by impregnating a hygroscopic thin fibrous material such as cotton,nonwoven fabric, or fabric cloth with collagen, and drying them, athrombus inducing material with a higher thrombus inducing ability canbe obtained. In addition, substrate may be dipped in a collagen solutioncontaining tissue thromboplastin and then dried to further enhance thethrombus-inducing ability thereof.

The thrombus inducing material can be selected depending on the innerdiameter of the thrombus formation chamber 10 and the monitoring object.When atherothrombosis such as myocardial infarction is provided for amodel, it is preferable to contain collagen solely or contain bothcollagen and tissue thromboplastin. In addition, it is more preferablethat a constriction portion may be formed on the channel in the thrombusformation chamber to provide the thrombus formation chamber with ashearing stress. Further, in the case of a thrombus examination ofcardiac cerebral infarction of cardiac origin or the like, in which athrombus may be transferred from another portion with blood flow andadhered to occlude the blood vessel of another portion, it is preferablethat a small amount of thrombus is previously adhered to the thrombusformation chamber 10 and provided as a thrombus inducing material,followed by monitoring the growth of thrombus formed thereon. In thecase of a thrombosis examination of the blood capillary, the inside ofthe channel in the thrombus formation chamber may be divided into aplurality of channels each having a narrowed width or thickness of 10 to30 μm. If the thrombus formation chamber 10 has a constriction portionwith 100 μm or less in width or thickness, the channel may be occludedwith a small amount of thrombus formed in such a constriction portion.Therefore, there is no need of using an additional thrombus inducingmaterial, so the thrombus formation can be monitored by a bloodcoagulation promoting agent or an anticoagulation releasing agent.Therefore, the present invention includes this constriction portion as athrombus inducing material.

The thrombus inducing material may be only coating with collagen or vWF.The thrombus formation chamber 10 at the coating portion may bepreferably constricted and provided for a constriction portion 14, sohigh shearing stress-induced platelet aggregation can be monitored. Inthe case of coating with collagen as a thrombus inducing material, it ispreferable that at least the substrate at a portion to be provided for abase is made of flat glass or plastic to obtain excellent adhesiveness.In addition, a portion on which the thrombus formation chamber 10 or aportion where the thrombus inducing material of the thrombus formationchamber 10 to be formed may be constructed as a detachable cassette.This case is preferable because the resulting thrombus can be easilywashed or observed, or the thrombus inducing material can be easilyexchanged with new one. In this case, the cassette may form aliquid-tight connection with a silicon rubber O-ring or the like.Preferably, the end of the cassette opposite to the end thereofconnecting to the inlet tube 11 of the thrombus formation chamber 10 maybe connected to a discharge tube 13 that permits discharge of the blood.Preferably, the discharge tube 13 may be divided with a cheese and thetip thereof may be then provided with a pressure gauge 41 such as adiaphragm-type one. On the other hand, the tip of the discharge tube 13is preferably connected to a storage container (not shown).

The inlet tube 11 connected to the thrombus formation chamber 10 can bemade using a transparent glass, a thermoplastic resin, or the like. Theend of the inlet tube 11, which is opposite to the other end thereofconnecting to the thrombus formation chamber 10, is connected to asyringe 20 that supplies blood. The syringe 20 is connected to a pump 30and a pressing means (not shown) so that the plunger of the syringe 20is pressed at predetermined pressure. The pump may be a general pumpcommercially available. Alternatively, the pump may be a syringe pumpconstructed by extruding the syringe with air at a constant pressure, orinverting the syringe so that the plunger is on the top side, andplacing a weight on the plunger.

The inlet tube 11 may be preferably divided by a cheese and the endthereof is preferably provided with a pressure gauge 40 such as adiaphragm-type one at a part of the inlet tube 11 near the thrombusformation chamber 10.

The blood in the syringe 20 is subjected to an anticoagulationtreatment. Examples of the anticoagulation treatment agent used in theanticoagulation treatment include sodium citrate or potassium citrate,sodium oxalate or potassium oxalate, Acid Citrate Dextrose (ACD), andethylenediaminetetraacetate (EDTA). Such an anticoagulation treatmentagent may be used in the form of powder, a lyophilized product, or asolution such as an aqueous solution. Among these anticoagulationagents, general 3.2% sodium citrate is preferable because it is easilyobtainable. In this case, one volume of the anticoagulation treatmentagent is preferably mixed with 9 volumes of blood.

In general, the whole blood or plasma without an anticoagulationtreatment agent is coagulated within several minutes. The coagulationcan be reduced or eliminated by the addition of a calcium chelator suchas citrate. In particular, it has been reported that citrate can inhibitthe agglutination and functions of prothrombinase and exogenous andintrinsic tenase.

The citrate-treated blood can be stored in liquid form for apredetermined period of time (for example, from several hours to severaldays) and processed into blood preparations such as a product ofcytapheresis, platelet-rich plasma, and platelet-poor plasma. Thecitrate-containing plasma can be stored at about −70° C. or lower for aprolonged period (from several months to several years). In the presentinvention, the whole blood and the plasma can be also used and, in thiscase, calcium or the like may be preferably added again.

However, in general, the whole blood or plasma added with calcium againis spontaneously coagulated due to contact activation in any of moststorage containers. In this case, the contact activation may occurwithin about 2 to 4 minutes. On this account, in the present invention,the blood newly prepared by a citrate treatment after blood sampling,the blood prepared by a citrate treatment after being frozen for storageand then defrosted, or platelet-containing plasma may be added with ananticoagulation releasing agent such as calcium, just after monitoringthrombus.

Other anticoagulation agents may include heparin, hirudin, hirulog(peptide of hirudin C-terminal region), aprotinin, antithrombinantibody, thrombin aptamer, maize-derived trypsin inhibitor (1977, J.Biol. Chem 252, 8105). These materials inhibit blood coagulation byinhibiting a coagulation cascade as a result of inhibiting a bloodcoagulation factor, so they will be sometimes referred to as“coagulation-factor inhibitors” in the specification of the presentinvention.

The blood for monitoring can be sampled by any method such as a methodin which coagulation-factor inhibitor is previously placed in a syringeor a vacuum blood-collecting vessel and the blood is then collected, ora method in which a coagulation-factor inhibitor is quickly added to theblood just after the blood sampling, to thereby obtain anticoagulatedblood.

Further, the blood is collected in a vacuum blood-collecting vesselcontaining a coagulation-factor inhibitor such as heparin, and thenheparinase and an anticoagulation treatment agent suitable for themonitoring object are added to degrade heparin so that hepatin isreplaced by an anticoagulation treatment agent suitable for themonitoring object. In addition, the blood is collected in a vacuumblood-collecting vessel containing citric acid and then added withcalcium chloride and a coagulation-factor inhibitor suitable for themonitoring object, such as a maize-derived trypsin inhibitor, or athrombin aptamer. Therefore, the anticoagulated blood can be collecteddepending on the monitoring object.

The drug tube 12 connected to the thrombus formation chamber 10 can bemade using a transparent glass, a thermoplastic resin, or the like. Theend of the drug tube 12, which is opposite to the other side thereofconnecting to the thrombus formation chamber 10, is connected to asyringe 21 for supplying a drug releasing the anticoagulation treatmentor a drug promoting blood coagulation. The syringe 21 may be connectedto a pump 31 and a pressing means (not shown) so that the plunger of thesyringe 21 can be pressed at predetermined pressure. The pump may be ageneral pump commercially available. Alternatively, the pump may be asyringe pump constructed by extruding the syringe with air at apredetermined pressure, or inverting the syringe so that the plunger ison the top side, and placing a weight on a plunger. The drug tube 12 isfilled with an anticoagulation releasing agent or a coagulationpromoting agent, as described later.

For carrying out monitoring the thrombus with the apparatus formonitoring thrombus formation of the present invention, for example, thesyringe 20 is filled with the whole blood or platelet plasma subjectedto an anticoagulation treatment with a sodium citrate treatment(solution A). The syringe 21 is filled with a drug that releases theanticoagulation treatment, such as a calcium chloride solution (solutionB). The solution A and the solution B are supplied into the inlet tube11 by the pumps 30 and 31, respectively, so the solution B can reach toa concentration of 5 to 20 mmol at which the coagulation cascade of thesolution A can be initiated. Subsequently, the solution A and thesolution B are mixed in the inlet tube 11, so that the mixture is flowninto the thrombus formation chamber 10. Further, for example, collagenor the like capable of inducing thrombus formation is previously appliedon a part of the inside of the thrombus formation chamber 10 to form athrombus inducing material. The thrombus formation chamber may be madeusing, for example, a transparent plastic tube. Thrombus formation canbe easily monitored by an apparatus for monitoring the blood passingthrough such a transparently visible thrombus formation chamber 10.

The monitoring of thrombus formation can be evaluated with visualobservation by flowing the blood for a predetermined period of timethrough a cell (thrombus formation chamber 10) treated with collagen andthen removing the blood therefrom. When the pump for feeding thesolution A and the solution B is air-driving, the solution A and thesolution B may be fed at constant pressure. Therefore, thrombusformation on the collagen can be monitored by a decrease in flow rate ofthe blood discharged from the discharge tube 13. Alternatively, when apressure gauge is mounted on the parts near the thrombus formationchamber 10 of the inlet tube 11 and the discharge tube 13, thrombusformation on the collagen can be monitored by observing a change ininner pressure of the thrombus formation chamber 10. Alternatively, itcan be observed under microscope by providing the thrombus formationchamber 10 with a thickness of 500 μm or less. In particular, thrombuswith platelet-rich plasma can be easily observed because it is highlyvisible and the thrombus formation thereof can be also observed by thenaked eyes. Further, it is also possible to fluorescently label theblood platelets and monitor the fluorescence thereof with a fluorescencemicroscope by the method described in Patent Document 1.

Examples of the drug for releasing the anticoagulation treatment by achelating agent such as citric acid include: calcium halides such ascalcium chloride, calcium bromide, and calcium iodide; inorganic calciumsalts such as calcium phosphate, calcium sulfate, calcium nitrate, andcalcium bicarbonate; and calcium salts of organic acids such as formicacid, acetic acid, propionic acid, butyric acid, alginic acid, lacticacid, gluconic acid, glyceric acid, and glycerophosphoric acid, whichare calcium compounds provided as free calcium donors.

The anticoagulation releasing agent can be selected and used dependingon the coagulation-factor inhibitor when an anticoagulation treatment iscarried out with a coagulation-factor inhibitor (anticoagulationtreatment agent). For example, as an anticoagulation releasing agentwhen the anticoagulation treatment with heparin is carried out,protamine, heparinase or antiheparin antibody can be used. As ananticoagulation releasing agent when anticoagulation treatments arecarried out with hirudin, hirulog, and aprotinin, anticoagulationreleasing agent such as antihirudin antibody, antihirulog antibody, andantiaprotinin antibody, respectively, can be used.

Examples of the anticoagulation releasing agent when an anticoagulationtreatment is carried out using antithrombin antibody as acoagulation-factor inhibitor include: a completely-inactivated thrombinsuch as a PPACK thrombin; the degradation fragment of thrombin, and asynthetic polypeptide containing an antibody-recognition epitope ofthrombin.

The antibodies used in the anticoagulation treatment or the release ofanticoagulation preferably include antibodies from which Fc domains areremoved by papainase or the like to minimize the effects thereof on thecomplement system or antibodies such as chicken egg antibodies withoutthe ability of activating the human complement system.

When a thrombin aptamer (Blood. 1993 Jun. 15; 81(12): 3271-6 or J MolBiol. 1997 Oct. 10; 272(5): 688-98.), which is a single-strand oligoDNA, is used as an anticoagulation treatment agent, a substance thatbinds to the thrombin aptamer and inhibits the functions thereof, suchas antisense DNA or antisense RNA, can be used as an anticoagulationreleasing agent. When two kinds of thrombin aptamers, one recognizingexosite I and the other recognizing exosite II, are used in combination,an extremely higher effect of the anticoagulation treatment can beobtained in comparison with the case in which each of them is solelyused. The antisense DNA used in this case may be one against part of thethrombin aptamer as long as it substantially inactivates theantithrombin function of the thrombin aptamer.

In addition, a fact that the thrombin aptamer and the antisense DNAthereof are effective as an anticoagulation treatment agent and ananticoagulation releasing agent, respectively, will be described withreference to reference examples as described below.

The coagulation system cannot be activated within several hours when ananticoagulation treatment is carried out with heparin. Thus, the bloodcan be stored for a long period of time in monitoring thrombusformation. However, for example, there is a case where theanticoagulation treatment is not suitable for examination of the bloodcollected from a patient subjected to heparin administration.

On the other hand, hirudin, hirulog, and antithrombin antibody areinhibitors for inhibiting the conversion of fibrinogen to fibrin byinhibiting thrombin which acts on the final stage of the coagulationsystem. However, a contact phase factor (such as pre-kallikrein or XIIfactor) of the coagulation cascade is gradually activated even in thecase of completely inhibiting thrombin, so the activation of theupstream of the coagulation cascade can occur. Therefore, there is acase that it is not suitable for a prolonged storage of the blood.

Also, aprotinin inhibits the activity of kallikrein in the contact phaseto delay the intrinsic blood coagulation cascade. However, thecoagulation cascade is gradually activated even under the inhibition ofkallikrein activity, so the blood is coagulated after several hours evenin the presence of aprotinin.

Therefore, in the monitoring of thrombus formation of the blood afterabout several tens of minutes, it is possible to monitor the thrombusformation by anticoagulation treatment with the inhibitor of thrombinand/or aprotinin. However, it may be unpreferable in the case ofrequiring a long time to start the monitoring or requiring a long timefor the monitoring.

When the thrombus formation is monitored after one hour or more from theblood sampling, an anticoagulation treatment may be carried out using athrombin aptamer in combination with a maize-derived trypsin inhibitor,hirudin, and aprotinin. Alternatively, a small amount of heparin, forexample, less than 1 unit/ml, which is a level that does not have agreat influence on the coagulation time, may be used in combination withan inhibitor of contact phase factor or a thrombin inhibitor.

An extremely higher effect of the anticoagulation treatment can beobtained when a thrombin aptamer is used in combination with two kindsof aptamers for exosite I and exosite II. Further, the antisense DNAs ofthe respective aptamers can release the anticoagulation treatment,immediately.

Also, an inhibitor of contact phase factor, such as a maize-derivedtrypsin inhibitor (XII factor inhibitor) or aprotinin (kallikreininhibitor), and an inhibitor of thrombin, such as a thrombin aptamer,are added to a sample (blood) in advance and then added with theantisense DNA of the thrombin aptamer (thrombin aptamer inhibitor) orthe like. Thus, the function of the thrombin inhibitor alone isinhibited to release the functional inhibition of thrombin, followed byquickly flowing the sample into the thrombus formation chamber. As aresult, for example, an extrinsic coagulation cascade can be activatedby a tissue factor (tissue thromboplastin) or the like, so the bloodcoagulation is promoted and the physiological thrombus formation can bemonitored.

Alternatively, anticoagulation treatments may be carried out with boththe maize-derived trypsin inhibitor and the anticoagulation treatmentagent which are capable of releasing an anticoagulation treatment, suchas a chelator (citric acid), heparin, or the like; and then a freecalcium donor such as calcium chloride, or heparinase may be allowed torelease the corresponding anticoagulation treatments, followed byallowing the blood to be flown into a thrombus forming chamber topromote blood coagulation by activation of the extrinsic coagulationcascade.

In this case, a thrombus inducing material may preferably contain anappropriate amount of a tissue factor (tissue thromboplastin).Particularly preferably, the thrombus inducing material may be coatedwith a mixture prepared by mixing collagen with tissue thromboplastinand then drying because thrombus formation is promoted only in thethrombus formation chamber. When a thrombus inducing material coatedwith a collagen solution containing a tissue factor (tissuethromboplastin) as an atheromatous thrombus model is used, monitoringthat reflects a pathological mechanism can be carried out.

In addition, the thrombus formation can be monitored by adding the freecalcium donor and the trypsin inhibitor to the blood subjected to theanticoagulation treatment with the calcium chelator such as citric acid,and quickly flowing the blood into the thrombus formation chamber, or byadding heparinase and trypsin inhibitor to the blood subjected to theanticoagulation treatment with heparin, and then quickly flowing theblood into the thrombus formation chamber. The method as describedabove, in which thrombus is monitored by mixing the anticoagulated bloodobtained using one kind or more kinds of anticoagulation treatmentagents with at least one anticoagulation treatment releasing agentcorresponding to the anticoagulation agent used and then quickly flowingthe blood into the thrombus formation chamber, may employ an apparatusfor monitoring thrombus formation as illustrated in FIG. 9 as describedbelow.

The above-mentioned thrombin inhibitors, such as hirudin and thrombinaptamers, synthesized low molecular inhibitors of contact phase factor,and anticoagulation treatment agents of protein are expensive comparedwith any of anticoagulation treatment agents such as citric acid andEDTA, which form chelates with calcium and the like. However, they donot change the concentrations of divalent metal ions such as calcium,magnesium, and zinc, so thrombus formation with the originalconcentrations of these ions in the patient's blood can be reflected tothe monitoring. Therefore, the monitoring that more reflects theclinical condition of the patient becomes possible.

Blood coagulation can be suppressed by the inhibition of a contact phasefactor such as activated factor XII or kallikrein. However, it has beenreported that the activation of XIIa and kallikrein does not contributevery much to the actual physiological thrombus or hemostasis. Actually,there is no finding of hemorrhage or the like even in a patient withcongenital deficiency of factor XII, pre-kallikrein, or the like at all.Particularly, in atherothrombosis such as myocardial infarction, it iswidely known that the coagulation system is activated by a tissue factordue to plaque caused by the arteriosclerosis. Factor XII can beactivated mainly by thrombin on the activated platelet. Therefore, inperforming an anticoagulation treatment, when an inhibitor forinhibiting the activation of factor XII or prekallikrein or an inhibitorfor inhibiting activated factor XII or kallikrein is used as ananticoagulation agent, there is no need to add the anticoagulationreleasing agent. By adding any substance that activates extrinsiccoagulation, such as a tissue factor (e.g., tissue thromboplastin), or athrombus inducing material to the blood instead of the anticoagulationtreatment agent or by adding the substance to the thrombus inducingmaterial, the coagulation system is activated in an alternative pathwaythat avoids the XII-factor activation and the kallikrein activation topromote blood coagulation, so thrombus can be monitored in the thrombusformation chamber.

As anticoagulation treatment agents, which can be used in monitoring thethrombus formation after promoting blood coagulation by the addition ofa substance that activates extrinsic coagulation, such as a tissuefactor without adding the anticoagulation releasing agent, synthesizedlow molecular inhibitors such as the Kallikrein inhibitor PKSI-527(Thromb Res 57: 889, 1990) and D-Phe-Arg-CK which is the activatedfactor XII inhibitor (Cal Biochem, Co., Ltd.) are exemplified. Inaddition, the protein inhibitors include antibodies againstcontact-phase protease in the coagulation cascade, such asanti-kallikrein antibody, anti-prekallikrein antibody, anti-coagulationfactor XII antibody, anti-activated coagulation factor XII antibody, andmaize-derived trypsin inhibitor.

For monitoring the thrombus formation after promoting blood coagulationby the addition of a substance that activates extrinsic coagulationwithout the addition of the anticoagulation releasing agent,particularly from the viewpoints of availability and anticoagulationability, it is preferable that the blood is once subjected to ananticoagulation treatment with a combination of citric acid andmaize-derived trypsin inhibitor or a combination of thrombin aptamer andmaize-derived trypsin inhibitor, and the blood is stored thereafter,because the degree of freedom of monitoring operation can be heightened.Further, just before thrombus monitoring, an anticoagulation treatmentwith a maize-derived trypsin inhibitor is maintained while anotheranticoagulation treatment is partially released by a free calcium donoror the antisense DNA of a thrombin aptamer. In this case, as a model ofatheromatous thrombus, it is preferable to activate extrinsiccoagulation by using a thrombus inducing material coated with collagenor collagen containing tissue thromboplastin to promote bloodcoagulation.

Containers used in the thrombus monitoring, such as a syringe forsupplying the blood and a vacuum blood-collecting vessel, are preferablycoated with heparin or with a material having antithrombus ability andblood compatibility, such as polyvinyl lactoamide (PVLA) orpoly-2-methoxyethyl acrylate (PMEA).

In an apparatus for monitoring thrombus formation according to anotherembodiment of the present invention, a tube and a thrombus formationchamber may be integrated with each other on a substrate by a finechannel such as a microchip.

FIG. 4 is a plane view of a main part of an apparatus for monitoringthrombus formation according to a second aspect of the presentinvention. In FIG. 4, a substrate 100 is grooved and a circuit is formedthereon. This embodiment is configured as follows. On a small substrate,a thrombus formation chamber as a main part of the apparatus formonitoring thrombus formation, an inlet tube which is connected to thethrombus formation chamber and through which blood is flown into thethrombus formation chamber, and a drug tube which is connected to theinlet tube and through which an anticoagulation releasing agent or adrug promoting blood coagulation is introduced into the thrombusformation chamber are integrated with each other and provided as amicrochip circuit. In this embodiment, parts other than the main part,which are integrated on the substrate are the same as those of the firstembodiment.

In FIG. 4, a substrate 100 is a main body of a microchip and thematerials thereof may be any of metal, glass, plastic, silicone, and thelike. In the light of observing a thrombus or the like, a transparentmaterial is preferable. In the light of forming a circuit, a plasticmaterial is preferable. Therefore, a transparent plastic material isparticularly preferable. When it is made of silicon resin, such aspolydimethyl siloxane (PDMS), the adhesiveness thereof is excellent.Thus, the circuit can be formed by contact bonding with a cover withoutadhesive or the like. When a substrate made of polystyrene is used, thechannel can be easily coated with PVLA and subjected to an antithrombustreatment. Further, PMEA may also allow a simple, effective antithrombustreatment (see Reference Example 4).

FIG. 5 is a plane view of a substrate to be provided as a cover of themicrochip which is overlapped and bonded to the substrate 100 of FIG. 4.A substrate 200 of FIG. 5 is a transparent slide glass or a plate or asheet that are formed of plastic or the like. A substrate 200 isprovided as a cover, and is overlapped and bonded to the substrate 100,so the circuit of the substrate can be formed of a thrombus formationchamber 110, an inlet tube 111, and a drug tube 112.

For providing the inner surface of the thrombus formation chamber 110with a thrombus inducing material 115, for example, collagen or the likemay be applied on the predetermined setting position of the substrate200. An apparatus for monitoring thrombus formation having a thrombusformation chamber can be obtained when the substrate 100 and thesubstrate 200 are bonded to each other by joining or fitting whiledirecting the collagen-applied surface of the substrate 200 inward.Because of simple application, collagen may be preferably applied to theflat substrate 200 having no groove. In addition, tissue thromboplastinis preferably applied after mixing with collagen, because a thrombusinducing material having a higher thrombus-inducing ability can beobtained. Further, for easily thrombus formation with applied collagen,the glass having the collagen-applied portion may be subjected to theadditional treatment, such as replacing the glass with a frosted glassor the like so that the surface area can be increased.

A connection part 100A, the connection part 100B, and the connectionpart 100C of the substrate 100 are connected to tubes (not shown) thatform parts of the inlet tube, the drug tube, and the discharge tube,respectively. Thus, the connection part 100A and the connection part100B are each connected to pumps (not shown) through the tubes. Theanticoagulated blood is injected from the connection part 100A, and theanticoagulation releasing agent corresponding to the anticoagulationtreatment agent is injected from the connection part 100B.

According to this embodiment, thrombus formation can be monitored withan extremely small amount of blood, thus the burden of the subject canbe made small. In addition, platelets attached on the substrate 200 fromthe substrate 200 side can be monitored by labeling the platelets with afluorescence reagent, such as mepacrine.

Further, the circuit 100D has a sealable end in which air is blocked.Thus, the circuit 100D is provided as a pressure gauge. The end of thecircuit 100D may be preferably provided with a regulation valve 100E forrelieving pressure or increasing sensitivity when the circuit 100D isprovided as a pressure gauge. The regulation valve 100E can be closed bya cap or an adhesion tape and the volume thereof can be changed withpacking such as resin depending on the required sensitivity. The circuit100D is formed narrow so as to prevent the blood from mixing with airand prevent air from escaping. A thickness of the circuit 100D dependson the material of the substrate 100, but is about 0.1 mm to 0.5 mm. Ifthe inner pressure of the inlet tube 111 of the apparatus for monitoringthrombus formation is increased by thrombus formation, the air in thecircuit 100D is compressed by the blood, so the anticoagulated blood canbe introduced into the circuit 100D by just that much. The innerpressure can be monitored by movement of the blood in the circuit 100Dat any time.

The adhesion of platelets to collagen, the activation of the coagulationsystem on the activated platelets, and the accumulation of activatedplatelets by the platelet agglutination can be monitored by theapparatus for monitoring thrombus formation and/or the method ofmonitoring thrombus formation of the present invention, respectively.

Further, for reproducing the thrombus formation of atherothrombosis,constriction portions 14 and 114 are formed on the thrombus formationchambers 10 and 110, respectively. Therefore, the monitoring, which alsoreflects a high shearing-stress creating platelet agglutination, can bemonitored.

FIG. 9 are schematic diagrams representing the main part of an apparatusfor monitoring thrombus formation according to a third embodiment of thepresent invention. FIG. 9 describe a blood coagulation inhibitor inlettube 322 and a pressure inlet tube 317, which mixes the bloodcoagulation inhibitor with the blood located downstream of the thrombusformation chamber. In this embodiment, the drug tube is not providedupper stream of the thrombus formation chamber.

According to the third embodiment, the blood collected after theaddition of anticoagulation treatment agents (e.g., citric acid andmaize-derived trypsin inhibitor) is further added with ananticoagulation releasing agent (e.g., free calcium donor) just beforethe measurement and then introduced into a syringe 320. Subsequently, aliquid for pressure-filling the blood, such as mineral oil, is pressedinto the syringe from a pressure inlet tube 317 connected to the syringe320, thereby extruding the blood to a microchip 300.

An increase in pressure exerted on the sample syringe 320 shows anoccluded state of the channel with thrombus formation, so the monitoringof thrombus formation with a change in pressure is particularly suitablefor the monitoring a model of strong atheromatous thrombus in which anobstructive thrombus is formed. The thrombus inducing material may be,but not specifically limited to, one prepared from collagen and a tissuefactor. For example, such a thrombus inducing material is effective tothe monitoring of thrombus when blood coagulation is promoted byactivating extrinsic coagulation.

At the time of pressure measurement, for a more correct measurement ofpressure in the channel with thrombus formation, as shown in FIG. 9, ablood coagulation inhibitor is introduced by the blood coagulationinhibitor inlet tube 322 and mixed with the blood located downstream ofthe thrombus formation chamber through a channel formed in the microchip300. Thus, the blood in the channel subsequent to the thrombus formationchamber can be prevented from thrombus formation. Therefore, such aconfiguration is preferable because a change in pressure in the channel,which is due to constriction or closure of the channel with thrombusformation in the thrombus formation chamber, can be specifically,exactly measured.

Such a blood coagulation inhibitor may be preferably, but notspecifically limited to, the anticoagulation treatment agent used in thepresent invention. In consideration of cost effectiveness andhandleability, it is preferable to suitably select any of those thatprevent blood coagulation by albuminoidal deformation, including analkali or acidic solution, alcohol, urea, and SDSs.

Here, in the embodiment illustrated in FIG. 9, the apparatus formonitoring thrombus formation of the present invention comprises asuction pump provided on a discharge tube 313 instead of a pressureinlet tube 317, so the apparatus can determine the degree of thrombusformation on the basis of a change in suction pressure (negativepressure). Such a configuration may lead to a more efficient measurementwhile preventing the occurrence of a situation that a liquid forpressure-feeding of mineral oil or the like are mixed with the blood ina contact region and the mixture is fed, when the amount of the blood issmall in the case of using a pump that indirectly extrudes the blood viaa liquid separated in a layer as described later.

Further, there is no need of using a closed container such as a syringeas a container (sample tank) for supplying the blood into the apparatusfor monitoring thrombus formation of the present invention. Thus, thecontainer may have no cover and may be opened to the air. As a result,the apparatus for monitoring thrombus formation can be simplified.Further, in the apparatus for monitoring thrombus formation of thepresent invention, the inner pressure of the cannel is negative. Thus,when the apparatus for monitoring thrombus formation of the presentinvention is prepared from a microchip, for example, even in the case ofan incomplete adhesion between the main body of the microchip and thecover of the microchip, there is no leak of the blood at all. In somecases, it may be directly used as an apparatus for monitoring thrombusformation only by fitting the main body of the microchip and the coverof the microchip together as long as the fitting is accurate.

FIG. 10 illustrates a further systematized thrombus-monitoring system A.

The thrombus-monitoring system A of FIG. 10 fills a micro-feeding pump330 with a liquid having a density smaller than the blood. Then, theliquid is pressed into a sample syringe 320 in which the blood is placedby a pressure inlet tube 317 and then layered on the blood, therebyextruding the blood into a microchip 300. The extruded blood is mixedwith anticoagulation releasing agent injected from a drug tube 312,followed by reaching to a thrombus formation chamber 311. A liquid forpumping the blood in a micro-feeding pump 330 may be any of oils andfats, such as liquid paraffin, mineral oil, and silicone oil, and anormal saline solution. In this way, it becomes possible to prevent boththe micro-feeding pump 330 and a pressure sensor 340 from being pollutedwith the blood by indirectly extruding the blood.

Further, such a pump for indirectly extruding the blood by the liquidbeing separated in a layer with respect to the blood can be employed inthe apparatus for monitoring thrombus formation according to any of theembodiments of the present invention.

Further, the pressure sensor 340 can determine the pressure applied onthe sample syringe 320 by the micro-feeding pump 330.

Further, in this thrombus-monitoring system A, the state of thrombusformation can be recognized in more detail by an image analysis. Inparticular, in the case of labeling platelets and white blood cells withquinacrine and then monitoring the adhesion and the agglutinationthereof to collagen, luminance per unit area due to the fluorescencecolor development is monitored by an image analysis. Thus, the resultsof the monitoring can be evaluated and stored as data. The imageanalysis can be carried out by processing an image captured by afluorescent stereoscopic microscope 308 with a CCD camera by a computer307 and representing the image on a display.

Therefore, in the thrombus-monitoring system A, it is possible todetermine a comprehensive state of thrombus formation from the resultsof the image analysis and a change in pressure applied on the samplesyringe 320.

Further, such a thrombus-monitoring system can be systematized using theapparatus for monitoring thrombus formation of any of the embodiments ofthe present invention.

FIG. 12 are schematic diagrams illustrating an apparatus for monitoringthrombus formation of a fourth embodiment of the present invention. Theapparatus for monitoring thrombus formation is provided with a camerafor the observation of thrombus formation.

In the apparatus for monitoring thrombus formation, for example, theblood which is anticoagulated with citric acid and a maize-derivedtrypsin inhibitor is added with an anticoagulation treatment releasingagent (e.g., free calcium donor) and quickly filled in a syringe 420.The syringe 420 is aspirated by a pump 414 connected to a discharge tube413 to allow the blood to pass through a thrombus formation chamber 411,thereby framing a thrombus. The degree of thrombus formation can bedetermined by the change in the suction pressure (negative pressure).

Also, for preventing the blood after passing through the thrombusformation chamber from coagulating to thereby clogging a discharge tube413, or from affecting on a pressure measurement, a thrombus formationinhibitor inlet tube 422 mixes a thrombus formation inhibitor with theblood after passing through the thrombus formation chamber.

Further, a camera 430 (e.g., CCD camera) is arranged under the microchip400 to take an image of the thrombus formation chamber, so the spaceabove the microchip can be effectively used. A rail 433 is able to movethe camera 430 back and forth and around under the thrombus formationchamber. By using the camera 430 which has a function of storing itsposition quantified as the X-axis and the Y-axis, it can take imageswhile regularly moving around a plurality of specific points. Asconfigured above, it is possible to monitor the process of thrombusformation with time over a wide area of the thrombus formation chamber411 even the magnification of the camera 430 is set to a high level.Further, an optical source 432 (e.g., LED) is preferably located aroundthe camera 430 as the optical source 432 can be simultaneously movedwhile keeping its positional relationship with the camera 430. It ispreferable that the optical source 432 is able to irradiate the light ata wavelength that can excite a specific fluorescent substance togetherwith white light depending on a fluorescent material to be provided asan imaging target, thereby allowing the excitation of variousfluorescent materials.

EXAMPLES

Hereinafter, the present invention will be described in detail withspecific examples. However, the present invention is not limited tothese examples.

Example 1

An apparatus for monitoring thrombus formation illustrated in FIG. 1 isused to fill a syringe 20 with 50 ml of a citrate-treated blood(solution A) in which 9 parts by volume of the blood immediately afterthe blood sample is mixed with 1 part by volume of 3.2% sodium citrate,and to fill a syringe 21 with 10 ml of 0.2M CaCl₂ (solution B). Thesyringes 20 and 21 are connected to transparent nylon tubes (inlet tube11 and drug tube 12) with an inner diameter of 3 mm. Both tubes arejoined together at a T-shaped joint (cheese) and then connected to apolycarbonate thrombus formation chamber 10 of 3 mm in inner diameterand 1 cm in length through a single nylon tube (inlet tube 11) of 3 mmin inner diameter and 3 cm in length. The thrombus formation chamber 10itself is constructed as a removable cassette. The joint portion betweenthe cassette and the nylon tube (inlet tube 11) is made liquid-tight viaan O-ring made of silicon rubber. A glass member is fixed on the insideof the cassette by an epoxy-based adhesive, thereby forming aconstriction portion 14. The constriction portion 14 is designed so thatthe most narrowed site of the constriction portion 14 may have an innerdiameter (the maximum gap between the constriction portion 14 and theinner wall) of 1.5 mm. In addition, the thrombus formation chamber 10 isconnected liquid-tight with a tube as a discharge tube 13 having thesame diameter and formed of the same material through an O-ring made ofsilicon rubber, and thus a thrombus monitoring apparatus 1 asillustrated in FIG. 1 is manufactured. Note that flange-type pressuregauges 40 and 41 are mounted on parts of the inlet tube 11 and thedischarge tube 13 near the thrombus formation chamber 10 via joints(cheeses), respectively. In addition, the glass material of theconstriction portion on the inner surface of the thrombus formationchamber 10 is prepared such that collagen is coated as a thrombusinducing material 15 on the glass constriction portion on the inside byimmersing in a 0.1 N acetic acid solution containing 1% insolublecollagen type I (manufactured by Wako Pure Chemical Industries, Ltd.)and then drying. The syringes 20 and 21 are inverted so that plunger areon the top side and weights are then placed on the plungers so as toallow the solution A and the solution B to be flown at 5 ml/min and at0.5 ml/min, respectively, to be syringe pumps.

When the solution A and the solution B are flown for 10 minutes, adifference between the pressure gauges 40 and 41 of the inlet tube 11and the discharge tube 13 is emerged after several minutes and such adifference is then increased with time. Simultaneously, it is confirmedthat the blood discharged from the discharge tube 13 is also graduallydecreased. When the flow of all solutions is completed, a physiologicalsaline solution is flown into the apparatus for monitoring thrombusformation 1 to wash the thrombus formation chamber 10. Thus, theformation of thrombus can be found in the thrombus formation chamber 10by visual observation.

Example 2

An apparatus for monitoring thrombus formation 1 is prepared andthrombus formation is then monitored in a manner similar to Example 1,except that the solution A of Example 1 is further added with anunfractionated heparin (prepared from pig mucous) at a concentration of1 mg/ml.

In this case, there is no pressure difference and no decrease in bloodflow occurred, and thrombus cannot be found in the thrombus formationchamber 10 by visual observation after washing with a physiologicalsaline solution.

Example 3

An apparatus for monitoring thrombus formation 1 was prepared in amanner similar to Example 1 with the exceptions that: a glass tube of 3mm in diameter and 5 cm in length was provided as a cassette typethrombus formation chamber 10; the syringes 20 and 21 were eachconnected to syringe pumps driven by electric motors; and, instead ofthe constriction portion 14 and the collagen coated as a thrombusinducing material 15 on the constriction portion 14, as shown in FIG. 2(a), silk of 1.5 cm in length immersed in collagen of Example 1 and airdried at 4° C. were attached by an adhesive as a thrombus inducingmaterial 15 on the inner surface of the thrombus formation chamber 10which is 5 mm inside from the connection part of the glass tube and theinlet tube 11.

After flowing the solution A in the syringe 20 at 3 ml/min and thesolution B in the syringe 21 at 0.3 ml/min for 15 minutes, the thrombusformation chamber 10 was detached and the inside thereof was then washedwith a physiological saline solution. As a result, visual observationconfirmed that thrombus 16 with a length of about 1 cm and a thicknessof about 1 mm in the form of a comet shape as shown in FIG. 2( b), whichseems to be formed from the downstream side of the silk as a startingpoint and extend to the downstream was attached on the inner surface ofthe glass tube. Here, it is considered that the influence of blood flowmay cause the thrombus in a comet shape.

The widest part of the thrombus had a width of about 3 mm.

Example 4

An apparatus for monitoring thrombus formation 1 was prepared in amanner similar to Example 3 with the exception that, instead of silk, asshown in FIG. 3( a), 50 μl of the blood without an anticoagulationtreatment was dropped on the inner surface of a glass tube by a Pasteurpipette and then left standing at room temperature for 15 minutes,thereby obtaining a disk-shaped thrombus with a diameter of about 2 mmas a thrombus inducing material 15.

In a manner similar to Example 3, after flowing the solution A and thesolution B, the thrombus formation chamber 10 was removed and then theinside thereof was washed with a physiological saline solution. As aresult, visual observation confirmed that thrombus 16 with a length ofabout 1 cm and a thickness of about 1 mm in the form of a comet shape asshown in FIG. 3( b) extending toward the downstream while wrappingaround thrombus as a thrombus inducing material 15 was attached on theinner surface of the glass tube. Here, it is considered that theinfluence of blood flow may cause the generated thrombus in a cometshape.

The widest part of the thrombus had a width of about 3 mm.

Example 5

An apparatus for monitoring thrombus formation 1 was prepared andthrombus formation was then monitored in a manner similar to Example 3with the exception that the solution A was further added with Argatroban(registered mark, manufactured by Daiichi Pharmaceutical Co., Ltd.) asan an anticoagulation treatment agent at a concentration of 0.1 mg/mlwith respect to the solution A of Example 3.

As a result, visual observation did not confirm the presence of thrombusin a glass tube after washing with the physiological saline solution.

Example 6

An apparatus 1 for monitoring thrombus formation is prepared andthrombus formation is then monitored in a manner similar to Example 3with the exceptions that the anticoagulated blood prepared by theaddition of hirudin at a concentration of 1 μg/ml with respect to thewhole blood is used as the solution A and antihirudin polyclonalantibody (manufactured by COSMO BIO CO., LTD.) dissolved at aconcentration of 1 mg/ml in a physiological saline solution with respectto the whole blood is used as the solution B.

In this case, almost the same thrombus formation is confirmed as that ofExample 3.

Example 7

There are prepared 10 ml of the whole blood (solution A) which wassubjected to an anticoagulation treatment by adding to the bloodimmediately after the blood sampling 10 μg/ml of aprotinin and 1 μg/mlof recombinant hirudin (manufactured by Wako Pure Chemical Industries,Ltd.); and 1 ml of a solution (solution B) which was prepared by addingan antihirudin polyclonal antibody (manufactured by COSMO BIO CO., LTD.)from which a Fc domain was removed by a papain-immobilized resin to1000-fold diluted physiological saline s solution of 1 vial/ml ofthromboplastin reagent (manufactured by Sysmex Corporation) as a drugfor promoting blood coagulation.

For manufacturing an apparatus for monitoring thrombus formation, asubstrate 100 shown in FIG. 4, which is made of polydimethyl siloxanewith a width of 40 mm, a length of 70 mm, and a thickness of 1.5 mm, anda substrate 200 shown in FIG. 5, which has a thickness of 1 mm and ismade of glass with the same dimensions, are used.

A circuit containing a thrombus formation chamber 110 is formed bycutting a groove with a depth of 0.5 mm on the surface of a substrate100 with a pattern shown in FIG. 4. The depth of the part of the grooveto be provided as an inlet tube 111 is 1 mm. The length of the circuit100D is 30 mm. A through hole of 1 mm in diameter is formed at the endof the groove and provided as a regulation value 100 E which is closedby an operable and closable cap. The thrombus formation chamber 110 hasa length of 30 mm and a width of 2.5 mm at the wider portion and aconstriction portion 114 having a width of 0.5 mm. Then, a through holewith a diameter of 1 mm is provided as each of the connection parts 100Aand 100B. In addition, a through hole with a diameter of 2.5 mm isprovided as a connection part 100C.

Collagen in the form of a band with a width of 10 mm is applied as athrombus inducing material 115 to the substrate 200 at a positioncovering the constriction portion 114 of the substrate 100 as shown inFIG. 5. The application of collagen is carried out such that therectangular masking tape corresponding to the constriction portion 114of the substrate 200 is attached and a silicone-based stripping agentSRX-211 (Dow Corning Toray Co., Ltd.) is then coated thereon.Subsequently, the masking tape is peeled off. Then, a solution ofcollagen type 1 (Wako Pure Chemical Industries, Ltd.) dissolved in 0.1Nacetic acid is dropped so as to be 1% thereof on the glass region freeof the stripping agent and then left standing for 1 hour at 25° C. Afterthat, collagen on the stripping agent layer is washed out with purifiedwater, and collagen is only applied to a rectangular glass regioncorresponding to the constriction portion 114, where the stripping agentis not applied.

Subsequently, the collagen-applied surface of the substrate 200 and thecircuit-formed surface of the substrate 100 are bonded togetherface-to-face.

Connection parts 100A and 100B are connected to silicone tubes having aninner diameter of 1 mm from the back surface (circuit-free side) of thesubstrate 100 and then connected to a 10-ml syringe filled with thesolution A and a 1-ml syringe filled with the solution B, respectively.These syringes are each attached to syringe pumps, respectively. Notethat another connection part 100C is connected to a silicon tube havingan inner diameter of 2.5 mm, which is provided as a discharge tube.

The solution A is injected from the connection part 100A at a flow rateof 0.3 ml/min. The solution B is injected from the connection part 100Bat a flow rate of 0.03 ml/min.

The solution A and the solution B are allowed to run from the connectionpart 100A and the connection part 100B for 5 minutes, respectively. Aphysiological saline solution is injected from the connection part 100Ato wash out the blood. As a result, thrombus formation is confirmedmainly on the region a and the region b of FIG. 6 on thecollagen-treated surface of the substrate 200.

Example 8

An apparatus for monitoring thrombus formation is prepared and thrombusformation is monitored in a manner similar to Example 7 with thefollowing exceptions: solution A is prepared such that the bloodimmediately after blood sampling is added with anti-factor XIIpolyclonal antibody (manufactured by COSMO BIO CO., LTD.), from which aFc domain is cut off by papain, at a concentration of 0.3 mg/ml andunfractionated heparin (pig origin, Wako Pure Chemical Industries, Ltd.)at a unit of 0.3; solution B is prepared by diluting a thromboplastinreagent (manufactured by Daiichi Pure Chemicals Co., Ltd.) 50 times; theapplication of collagen is carried out by pretreating thecollagen-applied region of a transparent polystyrene substrate 200 (1 mmin thickness) shown in FIG. 8 with the dropping of a solution preparedby dissolving potassium permanganate in concentrated sulfuric acid at aconcentration of 2 g/l, subjecting to reaction at 25° C. for 10 minutesfollowed by quickly washing with purified water, collagen is applied bydroppping a solution in which collagen type 1 (Wako Pure ChemicalIndustries, Ltd.) dissolved in 0.1N acetic acid is dissolved at aconcentration of 1% on the pretreated region, and leaving standing at25° C. for 1 hour followed by washing out with purified water; and asubstrate 100 without a constriction portion is used for the thrombusformation chamber 110 shown in FIG. 7. Note that the connection part100C of the substrate 100 is provided for a through hole of 1 mm indiameter and connected to a silicone tube of 1 mm in inner diameter as adischarge tube.

After flowing the solution A and the solution B for 10 minutes, aphysiological saline solution is injected from the connection part 100Ato wash out the blood, thereby confirming the attachment of red thrombuson the channel part of the collagen-applied region of the substrate 200.

Example 9

An apparatus for monitoring thrombus formation was prepared and thrombusformation was monitored in a manner similar to Example 7 with theexceptions that:

a solution A was prepared such that the blood immediately after theblood sampling was subjected to an anticoagulation treatment by theaddition of aprotinin at a concentration of 0.05 mg/ml and recombinanthirudin (manufactured by Wako Pure Chemical Industries, Ltd.) at aconcentration of 1 μg/ml and then added with quinacrine (Sigma Co.,Ltd.); and

the connection part 100B is closed by a cap and only the solution A isthen injected from the connection part 100A at a flow rate of 1 ml/min.

When observed with a fluorescence stereoscopic microscope focused on thecollagen-applied surface from the substrate 200 side, a fluorescencecolor development of green quinacrine was confirmed on the portion ofthe collagen-applied surface and such a region spreads out in as macularregion with time. After 10 minutes, the green fluorescence colordevelopment which may be due to thrombocytic adhesion was confirmed withthe most of the collagen-applied surface.

Example 10

An apparatus for monitoring thrombus formation was prepared and thrombusformation was monitored in a manner similar to Example 9, except thatsolution A was prepared without the addition of quinalin.

The blood was injected from the connection part 100A at a flow rate of 1ml/min. When the tube of the connection part 100C was pinched to closethe tube for 2 seconds during the injection, the blood was moved upinstantly to 20 mm from the branching point in the circuit 100Daccompanied by an increase in inner pressure. Even after opening theconnection part 100C, the blood does not move from the 20-mm point, sothe evidence of an increase in pressure could be confirmed.

Example 11

An apparatus for monitoring thrombus formation is prepared and thrombusformation is monitored in a manner similar to Example 8 with theexceptions that: the antithrombin polyclonal antibody (COSMO BIO CO.,LTD.) from which a Fc domain is removed by a papain is added so as to bein a concentration of 30 μg/ml instead of heparin of the solution A ofExample 8; and the solution B of Example 8 is further added with PPACKthrombin in a concentration of 5 mg/ml.

After flowing the solution A and the solution B for 15 minutes, aphysiological saline solution is injected from the connection part 100Ato wash out the blood. As a result, it is confirmed that red thrombus isattached on a channel portion of the collagen-treated region of thesubstrate 200.

Example 12

An apparatus for monitoring thrombus formation was prepared and thrombusformation was monitored in a manner similar to Example 7 with theexception that:

solution A was prepared such that 50 ml of the blood immediately afterthe blood sampling, which was added with 0.5 units/ml of heparin and 10μg/ml of aprotinin, was centrifuged at 800 rpm and platelet-rich plasma(PRP) was prepared from the supernatant, thereby obtaining the solutionA; and

a solution in which 1 vial/ml of a thromboplastin reagent (SysmexCorporation) was diluted 30 times with a physiological saline solutionwas used as solution B.

A thrombus formation of the constriction portion 114 was monitored for15 minutes using a stereoscopic microscope. A plurality of plateletclots attached on around the constriction portion 114 was confirmed. Itwas also monitored that the number of the clots increased while thesizes thereof also increased, with the platelet clots repeatingattaching and detaching.

Example 13

Thrombus formation was monitored by running solution A and solution B ina manner similar to Example 7 with the following exceptions:

solution A was prepared by adding 500 μg of erythrocyte collected fromthe centrifuged sediment to 20 ml of PRP of the solution A of Example12;

a solution in which 1 vial/ml of a thromboplastin reagent (SysmexCorporation) diluted 30 times with a physiological saline solution wasused as solution B;

a slide glass was prepared by coating the whole surface of the substrate200 with a silicone-based stripping agent SRX-211 (Dow Corning TorayCo., Ltd.); and

approximately 1 μl of the whole blood was attached on near the wideningend point of a widening portion successively extending to 2.5 mm inwidth, which is adjacent to upstream of the constriction portion 114 ofthe thrombus formation chamber 110 on the substrate 100, then the wholeblood was left standing at room temperature for 10 minutes to causecoagulation thereof, and a preliminary thrombus was formed to be used asa thrombus inducing material.

The thrombus-attached region was monitored by a stereoscopic microscopefor 15 minutes. The formation of a new red thrombus, which was locateddownstream from the preliminary thrombus as an origin, was confirmedafter about 5 minutes, gradually extended downstream up to 15 minutes,and the width thereof extended to 2.5 mm. It was confirmed that theblood flows along the formed thrombus or flows through the gaps betweenthe formed thrombus.

Example 14

An apparatus for monitoring thrombus formation was prepared and thrombusformation was monitored in a manner similar to Example 7 with thefollowing exceptions:

50 ml of blood immediately after the blood sampling added with anexosite I thrombin aptamer (GGTTGGTGTGGTTGG: SEQ ID NO. 1) at a finalconcentration of 5 μM and a maize-derived trypsin inhibitor (COSMO BIOCO., LTD.) at a final concentration of 30 μg/ml was centrifuged at 800rpm for 10 minutes, and platelet-rich plasma (PRP) was formed from thesupernatant and provided as the solution A;

antisense DNA (CCAACCACACCAACC: SEQ ID NO. 2) was dissolved in aphysiological saline solution so as to be 150 μM in concentration andprovided as the solution B; and

at the time of mounting a thrombus inhibitor 115, a solution, which wasprepared by mixing the collagen solution of Example 7 with a solutionprepared by dissolving 1 vial of thromboplastin reagent (SysmexCorporation) in 1 ml of purified water and then dialyzed at a ratio of5:1, was dropped onto a silicone-unapplied region and dried with air at4° C.

A thrombus formation of the constriction portion 114 was monitored for 5minutes using a stereoscopic microscope. A plurality of platelet clotsattached around the constriction portion 114 was continued. It was alsomonitored that the number of the clots increased while the sizes thereofalso increased, with the platelet clots repeating attaching anddetaching.

Example 15

An apparatus for monitoring thrombus formation was prepared and thrombusformation was monitored in a manner similar to Example 14 with thefollowing exceptions:

after preparing PRP by the same procedure as that of Example 14,antisense DNA (CCAACCACACCAACC: SEQ ID NO. 2) was added so as to be 15μM in concentration immediately before the measurement, therebyreleasing the anti-thrombin treatment and provided as the solution A;

the injection opening of the connection part 100B is closed in a mannersimilar to Example 9;

collagen type I (collagen reagent for coating a cell culture dish withcollagen, Type I-A stock solution, Cellmatrix Co., Ltd.) was usedinstead of the collagen solution of Example 14; and

only the solution A was flown at a flow rate of 0.2 ml/min. for 5minutes.

A thrombus formation of the constriction portion 114 was monitored usinga stereoscopic microscope. A plurality of platelet clots attached aroundthe constriction portion 114 was confirmed. It was also monitored thatthe number of the clots increased while the sizes thereof increased,with the platelet clots repeating attaching and detaching.

Example 16

An apparatus for monitoring thrombus formation was prepared in a mannersimilar to Example 7 with the following exceptions:

a groove of 0.1 mm in depth was formed on the surface of the substrateby cutting in a pattern shown in FIG. 7, while leaving partition wallssuch that convex rows each having a length of 1 mm and a width of 40 μmwere made stand on the whole surface of the bottom portion around thecenter of the thrombus formation chamber 110 with intervals of 40 μmtherebetween;

the injection opening of the connection part 100B was closed in a mannersimilar to Example 9; and

the substrate 200 was a simple slide glass without a surface treatment.The gap (channel) between the partition walls simulates a bloodcapillary.

The whole blood immediately after the blood sampling was subjected to ananticoagulation treatment by the addition of a maize-derived trypsininhibitor with a final concentration of 30 μg/ml and an exosite Ithrombin aptamer with a final concentration of 10 μM. Immediately beforemonitoring, antisense DNA of exosite I thrombin aptamer was added so asto be 30 μM in concentration, followed by injection from 100A at a flowrate of 0.05 ml/min for 5 minutes.

The thrombus formation in the gap between the partition walls wasmonitored using a stereoscopic microscope. As a result, the channelswere closed in sequence during the monitoring for 5 minutes. It wasmonitored that about half of the channels were closed by thrombusformation.

Example 17

A syringe-type solution-feeding system shown in FIG. 9 was used. Amicrochip 300, where the substrate and the cover of the microchip shownin FIG. 9(B) and FIG. 9(C), respectively, made of the same materials asthose of Example 7 were pressure-bonded, was used. The microchip 300comprises a blood coagulation inhibitor inlet tube 322 and a dischargetube 313 which were connected to the substrate 100 as in the case withthe discharge tube of Example 7. However, the grooves of the microchip300 of FIG. 9 are all formed of 200 μm in depth. Also, a channel wasformed such that the blood could be introduced into the channel from thesample syringe 320, while the channel had a width of 1 mm and a lengthof 40 mm and was connected to a thrombus formation chamber 311 composedof narrow channel having a width of 200 μm and a length of 10 mm and theblood coagulation inhibitor inlet tube 322. A solution was prepared suchthat 1 vial of the Sysmex Corporation PT reagent (tissue thromboplastin)was dissolved in 1 ml of purified water and then dialyzed in purifiedwater, the dialyzed product in purified water was mixed with collagentype I (manufactured by Nitta Gelatin Inc.) at a ratio of 1:1. Thesolution was applied to the position of the thrombus inducing material315 as shown in FIG. 9. After that, the applied portion was dried at 4°C. in air and thrombus formation chamber 311 was covered, which was usedas a thrombus inducing material 315. The blood was collected after theaddition of an anticoagulation treatment agent such that the finalconcentrations of the respective components in the syringe were 25 μm/mlfor maize-derived trypsin inhibitor, 10 μM for exosite I thrombinaptamer, 10 μM for exosite II thrombin aptamer(5′-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′: SEQ ID NO. 3). Immediately beforemonitoring, the blood thus collected was added with antisense DNAs withrespect to thrombin aptamers of exosite I and exosite II so that each ofthem would reach to a concentration of 40 μM. Then, a syringe (1 mlsyringe, manufactured by Terumo Corporation) was filled with the bloodand provided as the sample syringe 320 shown in FIG. 9. The My Flow(manufactured by Arbiotec Co., Ltd), which is a micro-feeding pumpcapable of monitoring the inner pressure of a liquid-feeding pump, wasconnected to a pressure inlet tube 317 shown in FIG. 9. Mineral oil waspressed into the microchip 300 from the top of the syringe 320 and theblood was pushed out into the microchip 300 at a flow rate of 50 μl/min,while the pressure exerted on the sample syringe 320 was monitored.

As a blood coagulation inhibitor, 1 M of Tris-HCl (pH 10) was flown fromthe blood coagulation inhibitor inlet tube 322 at a flow rate of 50μl/min. Pressure began to rise 6 minutes after starting the pressuremeasurement.

The pressure, repeating the changes thereof up and down due to themovement of thrombus (“control” shown in FIG. 13), rose up to 80 kPa.

Example 18

The blood sampling was carried out with the addition of ananticoagulation treatment agent in manner similar to Example 17.Subsequently, the same procedure as that of Example 17 was carried out,except that unfractionated heparin was added at a concentration of 1unit/ml. The measurement of pressure was then performed. The pressurebegan to rise 13 minutes after starting the measurement. The pressurerose up to about 10 kPa (“Heparin” shown in FIG. 13).

Example 19

The blood sampling was carried out with the addition of ananticoagulation treatment agent in manner similar to Example 17.Subsequently, the same procedure as that of Example 17 was carried out,except that ReoPro (LILLY Co., Ltd.) was added at a concentration of 50μg/ml. The measurement of pressure was then performed. An increase inpressure could not be confirmed in the measurement for 18 minutes(“ReoPro” shown in FIG. 13).

Example 20

The blood sampling was carried out with the addition of 3.2% citric acidso that the ratio of citric acid and the blood be 1:9. Further, theblood was added with a maize-derived trypsin inhibitor at aconcentration of 25 μg/ml to carry out an anticoagulation reaction.Immediately after adding calcium chloride to the anticoagulated blood sothat it should reach 15 mM with respect to the anticoagulated blood atthe time of monitoring thrombus formation, the blood was added to asyringe (1-ml syringe, manufactured by Terumo Corporation), followed byinflowing the blood into the same microchip as that of Example 17 at aflow rate of 40 μl/min. Subsequently, a change in pressure was measuredusing the same system as that of Example 17. The pressure began to rise15 minutes after starting the pressure measurement and it rose up toabout 60 kPa.

Example 21

The blood sampling was carried out with the addition of ananticoagulation treatment agent in manner similar to Example 20.Subsequently, the same procedure as that of Example 20 was carried out,except that heparin was added at concentrations of 0.2 units/ml, 0.5units/ml, and 1 unit/ml, respectively. The measurement of pressure wasthen performed. The results are shown in FIG. 14.

Example 22

The blood sampling was carried out with the addition of ananticoagulation treatment agent in manner similar to Example 20.Subsequently, the same procedure as that of Example 20 was carried out,except that ReoPro (LILLY Co., Ltd.) was added at a concentration of 2μg/ml. The measurement of pressure was then performed. The pressurebegan to rise 20 minutes after starting the pressure measurement and itrose up to about 15 kPa after 25 minutes (FIG. 15).

Example 23

A syringe-type solution-feeding system shown in FIG. 12 was used. Inaddition, a microchip 400, where the substrate 200 as a cover of themicrochip and the substrate 100 as a body of the microchip shown in FIG.12(B) and FIG. 12(C) were pressure-bonded with each other, was used. Adischarge tube 413 made of a Teflon (registered trademark) tube wasfilled with mineral oil and one end thereof was connected to themicrochip 400 through a connection tube 412 made of silicon rubber andthe other end thereof was connected to a suction pump 414 in which apressure sensor was installed. An inlet tube forming a channel of themicrochip 400 was connected to a syringe 420 through the connection tube412 made of silicone rubber. One end of the blood coagulation inhibitorinlet tube 422 made of Teflon (registered trademark) was connected to aliquid-feeding pump 423 and the other end thereof was connected to theterminal end of the thrombus formation chamber through the connectiontube 412 made of silicone rubber. In this way, the microchip 400 wasconnected to the discharge tube 413, the syringe 420, and the bloodcoagulation inhibitor inlet tube 422, and thus an apparatus B formonitoring thrombus formation was produced. A CCD camera 430 providedwith an illumination optical source 432 (LED) which was mounted on arail 433 and movable was set under the thrombus formation chamber.

All of the grooves in the microchips had a depth of 120 μm.

The channel in which the blood was introduced had a width of 800 μm anda length of 7 mm. The narrowed channel had a width of 200 μm and alength of 10 mm.

A solution was prepared such that 1 vial of PT reagent (tissuethromboplastin, Sysmex Corporation) was dissolved in 1 ml of purifiedwater and then dialyzed in purified water, the dialyzed product inpurified water was mixed with collagen type I (manufactured by NittaGelatin Inc.) at a ratio of 1:1. The solution was then applied to theregion of the substrate 200 of the microchip facing the channel of theconstriction portion on the surface to be pressure-bonded with thesubstrate 100 of the microchip. After that, the applied portion wasvacuum dried at 4° C. and the substrate 100 and the substrate 200 werethen pressure-bonded with each other, thereby providing a microchip 400having a thrombus formation chamber 411.

The blood sampling was carried out using a vacuum blood-collectingvessel in which sodium citrate provided as an anticoagulation agent wasenclosed. Immediately before introducing into the syringe 420, the bloodwas added with calcium chloride at a final concentration of 12.5 mM anda maize-derived trypsin inhibitor at a final concentration of 25 μg/ml.

The My Flow (manufactured by Arbiotec Co., Ltd), which is amicro-feeding pump capable of monitoring the inner pressure of aliquid-feeding pump, was connected to a discharge tube 413 as a pump414. Thus, the suction was performed at a rate of 15 μl/min while theinner pressure of the channel was monitored.

As a blood coagulation inhibitor, 1 M of Tris-HCl (pH 10) was flown fromthe blood coagulation inhibitor inlet tube 422 at a flow rate of 8μl/min. White thrombus formation was mainly observed after 4 minutesfrom the start of the pressure measurement, while a decrease in innerpressure was confirmed. In addition, it was confirmed that the innerpressure decreased 30 kPa after 10 minutes from the start.

Further, thrombus formation was observed by taking images while thecamera 430 was moved around the thrombus-forming region in the channel.The position where the thrombus formation was observed was memorized,and the camera 430 took images while regularly moving around a pluralityof specific points. As a result, growth and destruction of thrombus withtime was recorded with respect to a plurality of large thrombi.

Hereinafter, reference experiments will be performed as described belowto show the effectiveness of thrombin aptamers and the antisense DNAsthereof as anticoagulation treatment agents and anticoagulationreleasing agents, respectively.

Reference Experiment 1

The blood immediately after blood sampling, the blood prepared by addinga volume of one tenth of 300 μg/ml of a maize-derived trypsin inhibitorto the first blood, and the blood prepared by adding a thrombin aptamerthat recognizes exosite I to the second blood so as to be 5 μM inconcentration were provided. Then, for these three kinds of the blood,the time taken for coagulation were measured in Eppendorf tubes (made ofpolypropylene). The sample was reversed up side down every one minuteand the flowability thereof was confirmed. The blood free of additiveshowed a coagulation time of 9 minutes. In contrast, the blood addedwith the trypsin inhibitor showed a coagulation time of 22 minutes.Further, the blood added with the thrombin aptamer showed a coagulationtime of 62 minutes.

Further, when 5 μM of a thrombin aptamer that recognizes exosite 1 and 5μM of a thrombin aptamer that recognizes exosite II were used incombination, blood coagulation was not confirmed even after 3 hours evenin the case of the blood free of the trypsin inhibitor. Exosite Irecognition thrombin aptamer: 5′-GGTTGGTGTGGTTGG-3′ SEQ ID NO. 1,Exosite II recognition thrombin aptamer:5′-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3′ SEQ ID NO. 3.

Reference Experiment 2

The measurement of APTT was carried out on 200 μM of plasma with respectto each of sample A added with 10 μM of physiological saline solution,sample B added with 10 μM of 500-1 μM exosite I recognizing thrombinaptamer, and sample C added with 10 μl of a solution containing 500 μMexosite I recognizing thrombin aptamer and 1500 μM exosite I recognizingthrombin aptamer antisense DNA. The sample A was 44 seconds, the sampleB was 2 minutes or more, and the sample C was 45 seconds.

Reference Experiment 3

FIG. 11(A) is an analysis of a waveform of coagulation withthromboelastogram by storing the blood added with 10 μl of aptamers toexosite I and exosite II at room temperature for 15 minutes, and then byadding 40 μM of antisense DNAs of both aptamers. FIG. 11(B) is athromboelastogram waveform of the blood just after blood sampling.

As is evident from the results of FIG. 11(A) and FIG. 11(B), it is foundthat the addition of two kinds of thrombin aptamers allow the blood tobe stored and the antisense DNA can release the anticoagulationtreatment thereof.

From the results of Reference Experiments 1 to 3, it is evident that thecombination of the maize-derived trypsin inhibitor and the thrombinaptamer can effectively inhibit the coagulation of the whole blood andthat the anticoagulation effect of the thrombin aptamer can beinactivated by the antisense DNA to the thrombin aptamer.

Reference Experiment 4

Hereinafter, the efficiencies of PMEA coating to the storage of bloodand platelet adhesion on the substrate will be described.

(1) 1% PMEA-containing methanol solution prepared according to JP04-152952 A was applied to a 2.5-ml acryl resin container (innerdimensions: 1 cm×1 cm×4.5 cm) and then dried at 90° C. for 10 minutes.Subsequently, 760 μl of the blood subjected to an anticoagulationtreatment with 2% citric acid was added to each of a non-coatedcontainer and a PMEA-coated container, followed by aspirating with apipette every 5 minutes to confirm blood coagulation.

Consequently, the non-coated container showed an apparent increase inviscosity at 30 minutes and coagulation at 60 minutes. In contrast, thePMEA-coated container showed an apparent increase in viscosity at 35minutes, but the coagulation was prolonged to 90 minutes.

(2) The above PMEA was flatly coated on a transparent acryl plate. Itwas used instead of the collagen-coated plate of Example 9 (FIG. 8) andan adhesion experiment of quinacrine-labeled platelets and leucocytes inthe blood was carried out in a manner similar to Example 9.

As a result, apparent adhesion or agglutination of platelets andleucocytes was observed after 10 minutes from beginning of flowing theblood. In contrast, adhesion or agglutination of platelets andleucocytes on the PMEA-coated acryl plate was not observed even after 30minutes.

From the above results, in the apparatus for monitoring thrombusformation of the present invention, by coating the blood storagesyringes, the tube, the substrate, and the like with PMEA, thrombusformation can be suppressed in a place other than the thrombusmonitoring chamber. Therefore, it suggested that thrombus monitoringspecific to the thrombus monitoring chamber can be attained.

In the above description, the present invention has been described withreference to the preferred embodiments. However, the present inventionis not limited to the examples and embodiments described above, andvarious modifications may be applied as long as not departing from thegist of the present invention. For example, the pressure of the pump maybe set to be high and the inner diameter of the discharge tube may beset to be small. In this case, back pressure can be generated in theinlet tube and the discharge tube, so thrombus formation can bemonitored while controlling the flow volume of blood under the load ofinner pressure corresponding to the blood pressure. Further, the flowrate of blood at this time can be freely controlled by means of pumppressure and the degree of throttling the discharge tube.

INDUSTRIAL APPLICABILITY

The apparatus for monitoring thrombus formation and the method ofmonitoring thrombus formation of the present invention can be favorablyused in comprehensive evaluation of blood coagulation and plateletthrombus formation under a bloodstream-equivalent environment using thewhole blood or the plasma containing platelets, for evaluating theefficacy of an antithrombotic drug applied to a patient or the like.

1. A method for monitoring thrombus formation, comprising flowinganticoagulated blood into a thrombus formation chamber, in at least apart of which a thrombus inducing material that induces thrombusformation is provided, while releasing an anticoagulation treatment orpromoting blood coagulation, and monitoring thrombus formation.
 2. Themethod for monitoring thrombus formation according to claim 1, wherein:the anticoagulation treatment is a treatment with a calcium chelator;and the anticoagulation treatment is released by a free calcium donor.3. The method for monitoring thrombus formation according to claim 1,wherein: the anticoagulation treatment is a treatment with a thrombinaptamer; and the anticoagulation treatment is released by an antisenseDNA of the thrombin aptamer.
 4. The method for monitoring thrombusformation according to claim 1, wherein the thrombus formation ismonitored by flowing the anticoagulated blood into the thrombusformation chamber without releasing the anticoagulation treatment, whilepromoting blood coagulation.
 5. The method for monitoring thrombusformation according to claim 4, wherein the blood coagulation ispromoted by a tissue thromboplastin.
 6. The method for monitoringthrombus formation according to claim 1, wherein the anticoagulatedblood is obtained by using one or more kinds of anticoagulationtreatment agents, and said anticoagulation treatment is released by atleast one kind of anticoagulation treatment releasing agentcorresponding to the anticoagulation treatment agent used.
 7. The methodfor monitoring thrombus formation according to claim 6, wherein: saidanticoagulation treatment agent is an inhibitor for contact phase factorand a calcium chelator; and said anticoagulation treatment releasingagent is a free calcium donor.
 8. The method for monitoring thrombusformation according to claim 6, wherein: said anticoagulation treatmentagent is an inhibitor for contact phase factor and heparin; and saidanticoagulation treatment releasing agent is heparinase.
 9. The methodfor monitoring thrombus formation according to claim 6, wherein: saidanticoagulation treatment agent is an inhibitor for contact phase factorand a thrombin aptamer; and said anticoagulation treatment releasingagent is an antisense DNA of the thrombin aptamer.
 10. The method formonitoring thrombus formation according to claim 9, wherein said contactphase factor is blood coagulation XII factor or kallikrein.
 11. Themethod for monitoring thrombus formation according to claim 10, whereinsaid contact phase factor is blood coagulation XII factor, and aninhibitor for the blood coagulation XII factor is a maize-derivedtrypsin inhibitor.
 12. The method for monitoring thrombus formationaccording to claim 1, wherein said method determines pressure at a timeof inflow and/or outflow of blood in the thrombus formation chamber. 13.The method for monitoring thrombus formation according to claim 1,wherein said thrombus inducing material includes collagen and a tissuefactor.